Adjuvant like effect of vaccinia virus 14K protein: a case study with malaria vaccine based on the circumsporozoite protein

UNIVERSIDAD AUTÓNOMA DE MADRID

FACULTAD DE CIENCIAS

Departamento de Biología Molecular

Adjuvant like effect of vaccinia virus 14K protein: A case study with malaria

vaccine based on circumsporozoite protein.

Ph.D Thesis

Aneesh Vijayan Madrid, 2013

I, Aneesh Vijayan, declare that the thesis entitled ``Adjuvant like effect of vaccinia virus 14K protein: A case study with malaria vaccine based on circumsporozoite protein´´ and the work presented in it, carried out at CNB-CSIC under the guidance of Prof. Mariano Esteban, are my own. No part of this work has previously been submitted for a degree or any other qualification at this university or any other institution.

Parts of this work have been published as:

Adjuvant like effect of vaccinia virus 14K protein: A case study with malaria vaccine based on circumsporozoite protein. Vijayan et al; J Immunol 2012; 188:6407-6417.

Efecto adyuvante de la proteina A27 del virus vaccinia (14K) y sus aplicaciones en vacunas.

2011. Submitted to the Spanish Patent Office. Number: P201131854.

Aneesh Vijayan

Candidate.

Prof. Mariano Esteban Rodriguez. Prof. Jose Maria Requena.

Thesis Director. Thesis Tutor.

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Abbreviations.

Ab Antibody.

Ag Antigen.

APC Antigen Presenting Cell.

CD Cluster of differentiation.

CMI Cell Mediated Immunity.

CSP Circumsporozoite Protein.

CTL Cytotoxic T Lymphocyte.

DMEM Dulbecco´s Modified Eagle´s medium.

DNA Deoxyribonucleic Acid.

dsRNA Double stranded ribonucleic acid.

E.coli Escherichia coli.

EDTA Ethylene Diamine Tetraacetic Acid.

ELISA Enzyme Linked Immunosorbent Assay.

ELISPOT Enzyme Linked Immunosorbent Spot Assay.

GPI Glycophosphatidylinositol.

HBsAg Hepatitis B virus surface antigen.

HA Locus Hemagglutinin Locus.

i.d Intradermal.

IFN Interferon.

Ig Immunoglobulin.

IL Interleukin.

iNOS Inducible Nitric Oxide Synthases.

i.p Intraperitoneal.

ii IRF Interferon Regulatory Factor.

kDa Kilodalton.

LPS Lipopolysaccharide.

mAb Monoclonal Antibody.

MFI Mean Fluorescence Intensity.

MHC Major Histocompatibility Complex.

MSP-1 Merozoite Surface Protein 1.

MVA Modified Virus Ankara.

NFkB Nuclear Factor kappa-light-chain-enhancer of activated B cells.

NO Nitric Oxide.

NYVAC New York Vaccinia Virus.

OD Optical Density.

PBS Phosphate Buffer Saline.

PFU Plaque Forming Units.

rRNA Ribosomal RNA.

SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis.

STAT-1 Signal Transducer and Activator of Transcription 1.

TCA Trichloroacetic Acid.

T_CM Central Memory T-cells.

TEM Effector Memory T-cells.

TEMRA Terminally Differentiated Effector Memory T-cells.

TLR Toll Like Receptor.

TNF Tumor Necrosis Factor.

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List of Figures.

FIGURE 1:IMMUNOLOGY OF MALARIA.. ... 7

FIGURE 2:TYPES OF MALARIA VACCINES.. ... 12

FIGURE 3:DESIGN AND CONSTRUCTION OF PLASMID ENCODING CS-14K.. ... 40

FIGURE 4:BIOPHYSICAL AND BIOCHEMICAL PROPERTIES OF RECOMBINANT PROTEINS.. ... 42

FIGURE 5:14K FUSION PROTEIN FORMS OLIGOMERS/AGGREGATES IRRESPECTIVE OF ANTIGEN USED.. ... 43

FIGURE 6:SUCROSE GRADIENT ANALYSIS OF CS-14K PROTEIN. ... 44

FIGURE 7:KINETICS OF CS EXPRESSION BY MVA-CS. ... 45

FIGURE 8:LOCALIZATION OF PROTEINS IN MACROPHAGES.. ... 46

FIGURE 9:CS-14K FUSION PROTEIN DOES NOT INDUCE NEUTRALIZING ANTIBODIES AGAINST MVA.. ... 48

FIGURE 10:VACCINATION STRATEGY.. ... 49

FIGURE 11:STRONG INHIBITION OF LIVER STAGE PARASITE DEVELOPMENT BY CS-14K.. ... 50

FIGURE 12:CS-14K IMPROVES NO PRODUCTION IN MACROPHAGES.. ... 53

FIGURE 13: CS-14K PRIMING ELEVATES NO PRODUCTION IN MICE.. ... 54

FIGURE 14:CS-14K PROTEIN INDUCED CYTOKINE PRODUCTION IN MACROPHAGES.. ... 55

FIGURE 15:CHIMERIC PROTEIN ACTIVATES STAT-1 AND IRF-3 IN MACROPHAGES.. ... 56

FIGURE 16:PROTEINS INHIBIT NF-ΚB ACTIVATION.. ... 58

FIGURE 17:PROPOSED TLR SIGNALING MECHANISM BY CS-14K.. ... 59

FIGURE 18:CS-14K PRIMING IMPROVES THE QUALITY OF ANTIBODIES IN VIVO.. ... 61

FIGURE 19:CHIMERIC PROTEIN PRIMING GENERATES ELEVATED LEVELS OF HIGH AVIDITY ANTIBODIES.. ... 62

FIGURE 20:IFN-Γ ELISPOTASSAY.. ... 64

FIGURE 21:POLYFUNCTIONAL CD8⁺T-CELLS ARE PRODUCED IN CS-14K PRIMED MICE.. ... 66

FIGURE 22:EFFECTIVE CYTOKINE SECRETION BY CD8⁺T_EM IN CS-14K PRIMED MICE.. ... 68

FIGURE 23:POLYFUNCTIONAL CD8⁺T-CELLS PRODUCES MORE IFN-Γ AND TNF-Α PER CELL BASIS.. ... 69

List of Tables.

TABLE 2:CS-14K INDUCES STERILE PROTECTION. ... 51

TABLE 3:COMPARATIVE ANALYSIS OF IMMUNOGENICITY OF VARIOUS CS BASED VACCINES. ... 77

TABLE 4:COMPARISON OF IMMUNE CORRELATES DEFINED IN THIS THESIS WITH THOSE OBSERVED IN HUMANS. ... 82

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``A journey of thousand miles begins with a single step´´; this proverb is most apt for describing the end of a lifetime journey to obtain PhD. This thesis is a cumulative effort of various people who have contributed immensely and therefore I would like to take this opportunity to thank them for all their unbridled support for making this dream a reality.

I would like to start with the La Caixa foundation for the benevolent fellowship to carry out the research. Hope such philanthropic activities would further aid the development of science.

First and foremost I would like to thank Prof. Mariano Esteban, without whom I would have been lost. His endless support and encouragement made research a fascinating and enriching experience.

The innovative ideas, thoughtful advice and ingenious comments given during my tenure here helped me to achieve this goal. Thank you Mariano, for introducing me to the wonderful world of vaccines.

I would like to take this opportunity to express my sincere gratitude to Inés Merino, who was a big help during my initial days in the lab. Also I want to thank Ruben for all the help and the great stories about the countries visited. José, your jokes did make the time in the lab more fun filled and in addition to all your support. Thanks a lot guys.

I wholeheartedly thank all the members of Lab112 for their support and love through all these years. A special thanks to Carmen, for teaching me the basic techniques and guiding me. Your thoughtful comments and fruitful discussions have helped me a lot. Lucas, a good friend, with whom I collaborated on numerous occasions. You were indeed a friend in need. Juan, a jovial guy and a renowned mountaineer, your advice and help was indispensable. Ernesto, Mr. Dependable, always ready to help, best way to describe him. To be frank the Spanish summary of the thesis is his effort.

I thank Mauro for his wistful, engrossing talks and suggestions as well as for the light hearted jokes.

Bea and Susanna, I am grateful to them for all the help during my initial days in the lab. I would also like to thank Choguii for her supportive role and assistance. I also thank Ana for all the help rendered. I also like to extend my gratitude to Marivi and Suresh for all their support. Finally I

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also like to thank all the members of CNB in providing a congenial and stimulating atmosphere to learn.

I am extremely grateful to our collaborators at John Hopkins University, Prof. Fidel Zavala and Diego Espinosa for performing the challenge studies.

I thank all my friends in especially Satish and Tribhu for making my stay here and memorable one.

I also like to thank Abhimanyu and Gaurav for all the ``coffee moments´´ we had. All the wonderful times we all had here will be permanently etched in my mind.

No work can be completed without a source of inspiration, and my family was that source of inspiration. No amount of gratitude can ever repay the sacrifices they have made. Love you Acchan, Amma and Aadru. To them I dedicate this thesis.

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ABBREVIATIONS... 1

LIST OF FIGURES. ... III LIST OF TABLES. ... III ACKNOWLEDGEMENTS. ... IV ABSTRACT……….……….…IX INTRODUCTION ... 1

1.1 PLASMODIUM. ... 4

1.2 LIFE CYCLE OF MALARIA. ... 4

1.2.1 DEVELOPMENT INSIDE HOST. ... 4

1.2.2 DEVELOPMENT INSIDE VECTOR. ... 5

1.3 PATHOLOGY AND CLINICAL MANIFESTATION OF MALARIA. ... 5

1.4 MALARIA IMMUNOLOGY. ... 6

1.4.1 INNATE RESPONSES IN MALARIA. ... 7

1.4.2 ADAPTIVE RESPONSE IN MALARIA. ... 8

1.4.2.1 HUMORAL RESPONSES. ... 8

1.4.2.2 CELL MEDIATED IMMUNE RESPONSE. ... 9

1.5 MALARIA VACCINES. ... 11

1.5.1 PRE-ERYTHROCYTIC VACCINE. ... 12

1.5.1.1 CIRCUMSPOROZOITE PROTEIN:-A POTENT PRE-ERYTHROCYTIC ANTIGEN. ... 13

1.5.2 ERYTHROCYTIC VACCINE. ... 14

1.5.3 TRANSMISSION BLOCKING VACCINES... 15

1.6 VACCINE DESIGN STRATEGIES. ... 15

1.6.1 DNA VACCINE. ... 15

1.6.2 SUBUNIT VACCINE. ... 16

1.6.2.1 ADJUVANTS. ... 17

1.6.3 POXVIRAL VECTORS. ... 18

OBJECTIVES ... 21

MATERIALS & METHODS... 25

3.1 CELL LINES. ... 27

3.2 GENERATION OF RECOMBINANT VIRUS. ... 28

3.2.1 CONSTRUCTION OF VIRUS. ... 28

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3.2.2 PURIFICATION OF VIRUS. ... 28

3.3 GENERATION OF RECOMBINANT PLASMIDS. ... 28

3.4 RECOMBINANT PROTEIN PURIFICATION. ... 29

3.5 NEUTRALIZATION ASSAY. ... 30

3.6 NITRITE MEASUREMENT... 30

3.7 RNA EXTRACTION AND RT-PCR. ... 31

3.8 CONFOCAL MICROSCOPY. ... 31

3.9 ANIMALS AND IMMUNIZATION. ... 31

3.10 P.YOELII SPOROZOITE CHALLENGE... 32

3.11 ELISA AND ANTIBODY AVIDITY MEASUREMENT. ... 32

3.12 IFN-Γ ELISPOT ASSAY. ... 33

3.13 MULTIPARAMETER FLOW CYTOMETRY. ... 33

3.14 STATISTICAL ANALYSIS. ... 34

RESULTS ... 37

4.1 CHARACTERIZATION OF RECOMBINANT PROTEINS. ... 39

4.1.1 EXPRESSION AND PURIFICATION OF RECOMBINANT PROTEINS. ... 39

4.1.2 CHARACTERIZATION OF CS PROTEIN EXPRESSED BY MVA. ... 44

4.1.3 ANTIBODIES AGAINST CS-14K DO NOT NEUTRALIZE VACCINIA VIRUS. ... 47

4.2 CS-14K PROTEIN PRIMING CONFERS STERILE PROTECTION AGAINST MURINE MALARIA. ... 48

4.2.1 CS-14K ABROGATES THE LIVER STAGE DEVELOPMENT OF SPOROZOITES. ... 48

4.2.2 CS-14K PROVIDES STERILE PROTECTION AGAINST A LETHAL CHALLENGE. ... 51

4.3 IMMUNE CORRELATES OF PROTECTION. ... 52

4.3.1 CS-14K PROTEIN MODULATES INNATE IMMUNE RESPONSES IN MACROPHAGES. ... 52

4.3.1.1 NITRIC OXIDE. ... 52

4.3.1.2 MACROPHAGE SIGNALING AND CYTOKINE SECRETION. ... 54

4.4 CS-14K PROTEIN VACCINE IMPROVES THE QUALITY AND QUANTITY OF HUMORAL RESPONSE. ... 60

4.5 CS-14K GENERATES DURABLE AND POLYFUNCTIONAL CS SPECIFIC CD8⁺T-CELLS. ... 63

DISCUSSION ... 71

5.1 THEME OF CURRENT STUDY. ... 73

5.1.1 MURINE MALARIA:A PERFECT MODEL FOR DEVELOPING VACCINE CANDIDATES. ... 73

5.1.2 FUSION OF 14K TO AN ANTIGEN RESULTS IN OLIGOMERIZATION... 74

5.1.3 STERILE PROTECTION USING ADJUVANT FREE VACCINE. ... 75

5.1.4 HIGH QUALITY CS ANTIBODIES: HALLMARK OF A GOOD VACCINE. ... 77

5.1.5 CS SPECIFIC CD8+T-CELLS ROLE IN MALARIA: A BALANCE BETWEEN AMOUNT AND POLYFUNCTIONALITY. ... 79

5.1.6 ENGAGING THE INNATE IMMUNE SYSTEM. ... 80

CONCLUSION ... 83

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7.2 INTRODUCCIÒN. ... 90

7.2.1 MALARIA LA ENFERMEDAD. ... 90

7.2.2 LAS VACUNAS CONTRA LA MALARIA. ... 91

7.2.2.1 PROTEÍNA CIRCUMSPOROZOITO:POTENTE CANDIDATO VACUNAL PRE-ERITROCÍTICA. ... 91

7.3 RESULTADOS Y DISCUSIÓN. ... 92

7.4 CONCLUSIONES. ... 96

REFERENCES ... 99

APPENDIX ... 121

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ABSTRACT

Development of subunit vaccines for malaria that elicit a strong, long-term memory response is an intensive area of research, with the focus on improving the immunogenicity of a circumsporozoite (CS) protein-based vaccine. In this study, we found that a chimeric protein, formed by fusing vaccinia virus protein 14K (A27) to the CS of Plasmodium yoelii, induces strong effector memory CD8+ T cell responses in addition to high-affinity Abs when used as a priming agent in the absence of any adjuvant, followed by an attenuated vaccinia virus boost expressing CS in murine models. Moreover, priming with the chimeric protein improved the magnitude and polyfunctionality of cytokine-secreting CD8⁺ T cells. This fusion protein formed oligomers/aggregates that led to activation of STAT-1 and IFN regulatory factor-3 in human macrophages, indicating a type I IFN response, resulting in NO, IL-12, and IL-6 induction.

Furthermore, this vaccination regimen inhibited the liver stage development of the parasite, resulting in sterile protection. In summary, we propose a novel approach in designing CS based pre-erythrocytic vaccines against Plasmodium using the adjuvant-like effect of the immunogenic vaccinia virus protein 14K.

INTRODUCTION

3 Malaria, meaning ‘bad air’ in Italian due to its prevalence in marshy areas, continues to present a major public health challenge and burden on economic development in many countries.

Plasmodium falciparum, the main causative agent of malaria in humans, is known to cause approximately 225 million cases and about 781,000 deaths annually. Malaria continues to be a key factor involved in the mortality and morbidity among young children and mothers in African and Sub-Saharan areas (World Malaria Report, 2010). Areas which were previously declared malaria free are also under constant threat of resurgence due to changes in global weather and globalization. The situation has further worsened due to emergence of drug resistance parasites and ineffective vaccines. With several drugs in pipeline an effective vaccine is need of the hour in the fight against malaria.

To date vaccines have played an important role in the elimination of many diseases. However development of a malaria vaccine is curtailed by the ability of the parasite to deceive the immune system. Natural immunity to malaria is observed in endemic areas which also requires repeated exposure and takes considerable time to develop. However this kind of immunity ranges from partial to complete protection and is usually observed in older population. Therefore the development of an effective malaria vaccine is essential. Even though a large repertoire of antigens from various stages of malaria is available selecting a successful candidate is still an ongoing task. A successful vaccine should overcome several constraints such as it should provide long lasting protection irrespective of genetic variations that exists between human as well as parasite populations. Additionally it should be easy to produce and transport with minimal costs.

In addition the vaccine may incorporate antigens from different developmental stages of the parasite. With the advent of new technologies in vaccine development an effective malaria vaccine might not be a distant dream now.

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1.1 P

.

Plasmodium, the causative agent of malaria, belongs to the order coccidia a member of the Protista animal kingdom and Apicomplexa phylum . The family of Plasmodium is large with 172 different members infecting various eukaryotic species ranging from mammals, birds and reptiles. Only four members are known to infect humans viz; P.falciparum, P.vivax, P.malariae and P.ovale. Of these P.falciparum is the most prevalent with high mortality rates. Studies involving mouse malaria models, caused by P.yoelii and P.bergeii, helped us to understand malaria biology and are essential in development of various vaccines.

1.2 L

.

The vicious cycle of malaria requires two different species, one involving the host and other a vector.

1.2.1 DEVELOPMENT INSIDE HOST.

The host cycle is initiated with the blood meal by the infectious female Anopheles mosquito.

During the meal the mosquito injects the sporozoites, contained in its salivary gland, into the subcutaneous tissue of the host. Recent studies has shown that not all sporozoites inoculated enters the bloodstream, some of them migrate into the draining lymph nodes (Chakravarty et al., 2007). The sporozoites are then transiently circulated in the blood before it home to the liver.

The parasites pass through a number of hepatocytes before it establishes an infection, aided by CD81 marker on hepatocytes, and then replicates (Silvie et al., 2003). The whole process takes approximately 30 minutes and is known as the pre-erythrocytic stage. Inside hepatocytes the parasite undergoes several cycles of asexual replication to produce exoerythrocytic schizonts, each of which contains several thousands of merozoites; this typically takes about 2 to 15 days.

These exoerythrocytic schizonts ruptures releasing the infectious merozoites which then infects the circulating RBC’s initiating the erythrocytic stage. During this stage the ring shaped parasite develops in a parasitophorus vacuole into trophozoites. This stage lasts for 48 to 72 hours during which the infected RBC’s harboring the merozoites ruptures releasing them which then infect more RBC’s. This stage is responsible for the clinical manifestation of the disease. Some

INTRODUCTION

5 merozoites escape and undergo sexual replication forming the male and female gametes. Several factors are known to promote gametogenesis. These gametes are taken up by the mosquito during the blood meal.

1.2.2 DEVELOPMENT INSIDE VECTOR.

Among the different species of mosquitoes only the female sex of the Anopheles genus transmits malaria in humans. The vector is not just a repertoire for harboring the male and female gametes taken up from the host but its biological variations effects the development and transmission of the parasites (Beier, 1998; Chugh et al., 2011). During a blood meal the female mosquito ingests the sexual stage of the parasites which then undergoes fertilization to produce a zygote which differentiates into motile ookinete. Fertilization occurs in the midgut of the mosquito following which the ookinete penetrates through the epithelial cell wall of the midgut to form oocyst. The oocyst undergoes meiosis to produce sporozoites which then migrates into the salivary gland.

These sporozoites are then inoculated into the host during the blood meal.

1.3 P

.

Pathological symptoms of malaria are associated with the asexual replication of the parasite during the erythrocytic stages. The clinical manifestation of the disease is linked to the frequent bouts of fever linked to the rupture of erythrocytes and the subsequent release of merozoites.

Other symptoms includes myalgia, nausea etc and in severe cases leads to seizures, coma, renal failure, jaundice etc. Studies have elucidated the role of toxins and other factors that when released during the rupture of erythrocytes results in the activation of pro-inflammatory cytokines such as IL-12, IL-1, TNF-α etc. The main culprit is GPI (Glycosyl Phosphatidyl Inositol), a glycolipid (Arrighi and Faye, 2010). Evidences suggest that purified GPI from parasite is able to stimulate a pro-inflammatory response (Kamena et al., 2008).

In patients with severe malaria comparatively high levels of parasitemia has been reported (Anstey and Price, 2007). This usually leads to an increase in the expression of adhesion molecules such as CD36, ICAM-1 etc by vascular endothelial cells which aids the sequestration

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of infected RBC’s into post capillary venules effecting the oxygen supply (Cserti-Gazdewich et al., 2012; Serghides et al., 2003). This leads to organ failure and when the organ involved is the brain (cerebral malaria) it leads to coma and eventually death. The main parasitic protein involved in this process is called PfEMP-1 (P.falciparumErythrocyte Membrane protein) (Miller et al., 2002). Another complication associated with high parasitemia is the binding of sequestered iRBC’s with iRBC’s or uninfected ones to, this is known as rosetting, which often hampers the development of immunity against malaria (Vigan-Womas et al., 2008).

1.4 M

I

.

Immunity to malaria is a slow process and is influenced by factors such as age, area of transmission and the frequency of exposure to the parasite. So naturally acquired immunity against malaria occurs in only malaria endemic areas and in older generation through continuous exposure. However such immunity is known to wane faster and in most cases is unable to prevent disease severity (Doolan et al., 2009; Greenwood, 1999). There is a fine line between immunity and immunopathology in malaria. The presentation of large repertoire of antigens further confuses the immune system in mounting an appropriate response (Fig 1). An important question which continues to baffle malaria immunologists is the requirement of a chronic infection in order to maintain natural immunity. Even though studies have shown the importance of both innate and adaptive responses, we are yet to define the correlates of protection in controlling malaria.

INTRODUCTION

7 Figure 1: Immunology of malaria. A schematic representation of immune responses directed during various stages of parasite invasion (Riley and Stewart, 2013).

1.4.1 INNATE RESPONSES IN MALARIA.

Innate immune responses are carried out by a variety of cells including macrophages, monocytes, NK cells, granulocytes etc and generally acts as the first line of defense. They are also essential in shaping up the adaptive response via presentation of antigens and production of cytokines. An early innate response involving type I IFN response is essential in nipping the infection at the pre-erythrocytic stage (Arnold et al., 2010; Nussler et al., 1991; Shio et al., 2010). Like many parasitic diseases, TLR´s play a significant role in driving the immune response during malaria.

Role of TLR´s are controversial due to its pros and cons during the infection (Coban et al., 2007;

Franklin et al., 2011).

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Macrophages, the sentinels of the innate immune response, have tremendous parasiticidal activity which involves direct and indirect actions. Several studies has emphasized the direct role of macrophages by phagocytosing iRBC´s which reduces the initial parasitemia by complement system (Silver et al., 2010) or ADCI (Langhorne et al., 2008). Additionally, recent studies have also shown macrophage dependent secretion of cytokines and reactive oxygen species aids not only in controlling the initial parasitemia but also helps to shape long term memory immune response. However recent studies have shown that like in Leishmaniasis, the sporozoite has also evolved mechanisms to impair the functions of macrophages. Excessive activation of macrophages results cytokine storm which does more harm than protection (Perkins et al., 2011).

The abnormalities in iRBC´s results in rapid phagocytosis by the macrophages of both iRBC´s and the uninfected ones leading to severe anemia (SMA) (Kai and Roberts, 2008).

Thus the role played by macrophages in malaria is indispensable and a fine line exists between its role in protection or pathology.

1.4.2 ADAPTIVE RESPONSE IN MALARIA.

Development of adaptive response against malaria via natural infection is a slow process and the major mediators involved are the antibodies and members of cell mediated immune system.

1.4.2.1 Humoral responses.

The role antibodies play in malaria is quite indispensable considering the fact that they are one of the early mediators of protection in malaria. Seminal studies based on transfer of purified antibodies from immune donors to naïve individuals have shown the role played by antibodies in building malaria immunity (Ak et al., 1993; Cohen et al., 1961). The humoral response to different antigens during each stage of parasite development whether within host or vector is known to provide protection. During the pre-erythrocytic stages antibody developed against circumsporozoite protein (CSP) (Tapchaisri et al., 1985) and sporozoite surface protein (SSP-2) (Rogers et al., 1992) prevents infection. In the next stage i.e. erythrocytic level, the iRBC´s express on its surface variety of highly polymorphic parasitic protein known as VSA (Variant Surface Antigens). Antibodies against VSA´s are known to protect individuals from severe

INTRODUCTION

9 malaria (Chan et al., 2012). Additionally, antibodies against Pfemp-1 protein of malaria are also known to reduce the incidence of the disease especially in people residing in malaria endemic areas. Vaccines based on MSP-1 (Merozoite Surface Protein) capable of inducing antibodies are also gaining importance due to its effectiveness in reducing parasitemia. Another class of antibodies targeting the sexual stages of parasite, acting as a transmission blocking agents, are also of critical importance (Carter and Mendis, 1991). However not in all cases seropositivity against malaria antigens results in lifelong antibody titers. This could be explained by the defective development of B-cells (Dorfman et al., 2005).

Quality of the antibodies influences the outcome of the disease. Enhanced levels of cytophilic antibodies such as IgG1 and IgG3 have been reported in providing protection against malaria (Duah et al., 2010; Elliott et al., 2005). Antibodies produced against various surface antigens of merozoites were able to activate monocytes via FcRγII to release TNF and other mediators eliminating the infected erythrocytes in process known as antibody dependent cell mediated inhibition (ADCI) (Jafarshad et al., 2007). Antibodies are also known to neutralize the infection of RBC´s by merozoites (Williams et al., 2012). The role played by IgE antibodies in malaria is confounding due to reports which suggest its role in protection (Bereczky et al., 2004) as well as pathology (Perlmann et al., 1997).

An insight into the development of humoral response in malaria and the potential antigens could open up whole new aspects in our understanding of malaria and develop effective vaccines.

1.4.2.2 Cell mediated immune response.

In a seminal study the importance of cell mediated response in controlling malaria was first proven in animals that were thymectomized, making them more susceptible to infection (Brown et al., 1968). Protection in T-cells depleted animals by adoptive transfer of different antigen specific CD4⁺ and CD8⁺ T-cells further bolstered the importance of CMI in controlling malaria (Chakravarty et al., 2008; Stephens et al., 2005; Stephens and Langhorne, 2010).

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CD8⁺ T-cells:

Studies from animals vaccinated with multiple doses of γ-irradiated sporozoites, the only vaccine which induced sterile protection in animals, is mainly mediated by IFN-γ secreting CD8⁺ T-cells (Malik et al., 1991). Evidence for the role of HLA Class I mediated CD8⁺ protection against severe malaria was shown in children carrying the HLA-B53 MHC I allele in Gambia (Hill et al., 1992). Though most of our understanding about the role of CD8⁺ cells in malaria was restricted to the pre-erythrocytic stage, recent advances have made it possible to evaluate the responses in the liver stage as well. Studies have illustrated that an effective CD8⁺ response is influenced by IL-12 dependent production of IFN-γ, TNF-α and NO (Stevenson et al., 1995). CD8⁺ responses known to provide protection during the pre-erythrocytic and liver stages, cannot mediate protection during the blood stages. Studies based on CD8 transfer and in ß2 microglobulin deficient mice demonstrated that CD8 T-cells are not effective against the blood stages (van der Heyde et al., 1993). Initial studies considered liver to be the homing organ for anti-malaria CD8⁺ T-cells, however in a pioneering work carried out by Zavala and colleagues showed that in fact the skin draining lymph nodes are the primary sites for the induction of CD8⁺ T-cells against the liver stages (Chakravarty et al., 2007). Therefore in order to design effective CD8 T-cell based vaccine against malaria an important point to be considered is the skin immunity.

CD4⁺ T-cells:

Accumulating evidence supports the role of CD4⁺ T-cell response in regulating parasitemia or elimination of parasites (Meding and Langhorne, 1991; Shibui et al., 2009). Other than the direct effector function these cells are also important in the maintenance and survival of malaria specific CD8⁺ T-cells (Overstreet et al., 2011). Even the most advanced malaria vaccine, RTS,S/AS01E a protein in adjuvant vaccine, mediates protection via memory CD4⁺ T-cells secreting IFN-γ and TNF-α (Lumsden et al., 2011). In addition, effector CD4⁺ T-cells producing IL-10 via IL-27 dependent path is known to protect from the severe immunopathology associated with malaria (Freitas do Rosario et al., 2012). Furthermore in malaria endemic areas FOXP₃^- CD45RO⁺CD4⁺ T-cells were linked to reduced pathological condition seen in severe malaria

INTRODUCTION

11 (Walther et al., 2009). These cells were in fact independent of TCR stimulation but rather dependent on cytokines such as IL-10, TGF-ß and IL-2 (Scholzen et al., 2009).

Thus a strong balanced CMI response along with a quality humoral response is required to mount an effective strike against parasite. Therefore this concept should always pave the way while developing effective vaccines.

1.5 M

V

.

Vaccines are an essential tool in the armory for a fight against malaria. Considering the evolution of insecticide and drug resistance parasites, development of an effective vaccine is imperative.

Development of a cost-effective vaccine is obscured because of the confounding ability of the parasite to manipulate the host immune system. In spite of the wide array of the antigens available, which are expressed by the parasite at various stages in its life cycle, pointing down a specific antigen has been quite intricate. Depending on the life cycle of the parasite, vaccines developed can be classified under three classes (Fig 2); viz (1). Pre-erythrocytic Vaccines (2).

Erythrocytic Vaccines and (3). Transmission Blocking Vaccines. Since the most ideal vaccine should be the one that prevents the onset of clinical symptoms, we will be discussing more about the pre-erythrocytic vaccines.

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Figure 2: Types of malaria vaccines. Classification of malaria vaccines based on its action at different stages (Adapted from Malaria Path Initiative website).

1.5.1 PRE-ERYTHROCYTIC VACCINE.

Pre-erythrocytic vaccines involve antigens expressed right from sporozoite inoculation till the liver stage development. Therefore these vaccines prevent the occurrence of the disease.

However an important drawback with this vaccine is that the window to mount an attack against the sporozoite is very short before it homes to the liver. In addition it has to overcome the problems associated with the polymorphic nature of T-cell epitopes. Even though the main protagonists during this stage are the antibodies, CD8⁺ T-cells are also known to play a significant role during the liver stage. Liver being an immuno-tolerant organ, generating appropriate responses is a big hurdle. However there are overwhelmingly adequate data which supports the development of IFN-γ and TNF-α secreting CD8⁺ T-cell in liver, especially in the γ- irradiated sporozoite vaccine model (Epstein et al., 2011). Activation of macrophages, by the

INTRODUCTION

13 IFN-γ secreted by CD8⁺ T-cell, helps in phagocytosis and also increases NO within hepatocytes (Seguin et al., 1994).

RTS,S vaccine, the current advanced vaccine against malaria, is a pre-erythrocytic vaccine. This vaccine comprises the CS protein fused with HbSAg in excess of HbSAg to form VLP´s (Stoute et al., 1997). Main mediators of protection in this model are CD4⁺ T-cells in addition to antibodies (Lumsden et al., 2011). Even the golden model for malaria vaccines, γ-irradiated sporozoites, also promotes the development of intra-hepatic CD8⁺ T-cells. The latest data from RTS,S phase III clinical trials shows an overall efficacy of 16.8% in children after 4 years (Olotu et al., 2013). The dismal performance of the vaccine can be attributed to the waning immunity especially the antibody titers of CS with time. Therefore better pre-erythrocytic vaccines could prove beneficial in controlling malaria.

1.5.1.1 Circumsporozoite protein: - A potent pre-erythrocytic antigen.

Most of the pre-erythrocytic stage vaccines target the proteins that are expressed on the surface of the sporozoite. The most valued antigens that are targeted during this stage are the CS protein and TRAP protein. However a vaccine based on CS protein was found to be more promising and has successfully entered the phase III clinical trials in the form of RTS,S.

CS protein is a major monomeric protein found on the surface of the sporozoites. The importance of CS protein as a major vaccine candidate was first reported by Nussenzweig and colleagues (Nussenzweig and Nussenzweig, 1985). Most of the protection in γ-irradiated and genetically attenuated sporozoite vaccine model was mediated by CS specific humoral and CMI responses (Kumar et al., 2009). The protein maintains similar structural, biochemical and immunological properties across different species of Plasmodium. The central domain of all Plasmodium species contains a repeat region which contains immunodominant repeat regions of B and T-cell epitopes (Lal et al., 1987). This region is usually flanked by conserved region I at the N-end and region III and region II+ at the C-end. Binding of sporozoites to the GAG chains of HSPG of hepatocytes is mediated by the region II of CS protein (Pinzon-Ortiz et al., 2001). Native CS has rod like structure which in anchored to the surface of the sporozoites by a GPI motif found at the C

14

terminal region (Plassmeyer et al., 2009). In a seminal study it was shown that CS binds to the ribosomes disrupting protein synthesis in infected hepatocytes (Frevert et al., 1998). CS protein also aids in the survival of sporozoites in hepatocytes by blocking the translocation of p65 into the nucleus thereby blocking the NFkB pathway. In addition it also promotes the expression of various genes vital in the metabolic process to allow the parasite to thrive inside the infected cells (Singh et al., 2007). Inspite of certain disadvantages vaccines based on CS protein are continuingly making progress.

1.5.2 ERYTHROCYTIC VACCINE.

Vaccines designed against this stage targets the blood stage antigens. Immune response generated at his stage can only reduce the intensity of the disease by reducing the parasitemia.

Antigens expressed at this stage undergo rapid mutations and are highly polymorphic to overcome the pressure from the immune responses. Due to the lack of MHC I molecules on RBC´s an ideal erythrocytic vaccine candidate must induce high titer, high avidity antibodies.

These antibodies, mainly directed against merozoites, prevent merozoites from infecting new RBC. However reports of malaria specific T-cell responses during this stage are also reported.

This is mainly due the cross presentation of the antigens by professional APC (Miyakoda et al., 2008). In fact IFN-γ secreting CD8⁺ T-cells against blood stage antigens were reported in patients in malaria endemic areas (Sinigaglia et al., 1985).

MSP is the most widely studied blood stage protein. Abundance of this immunogenic protein on the surface of the merozoites makes it an ideal candidate for vaccine development. Clinical trials based on this antigen have progressed to phase II (Schwartz et al., 2012). MSP₁₉, a cleavage product of proteolysis, is highly conserved and is the main vaccine candidate. Rapid sero- conversion associated with this vaccine results in high titer antibodies capable of neutralizing merozoites (Hirunpetcharat et al., 1997). Sera transfer from vaccinated mice to immunocompromised mice did not result in protection even though a delay in parasitemia was observed suggesting that other factors also play an important role in this vaccine model. Some of the other blood stage vaccine candidates include AMA-1, PfEMP-1, RESA etc.

INTRODUCTION

15 Thus a blood stage vaccine can ameliorate the symptoms associated with malaria the individual still remains susceptible to infection. Therefore sterile protection with these vaccines is not possible which is reflected by reduced number of candidates entering clinical trials.

1.5.3 TRANSMISSION BLOCKING VACCINES.

Vaccines belonging to this category mainly target the sexual stage antigens of the parasite. The main goal with this vaccine is to reduce the disease morbidity in an area by preventing the development of the parasite in the vector thereby preventing new infections. Mostly antibody mediates this kind of protection wherein during a blood meal the antibodies taken up by the vector hampers with the proper development of the gametocytes in the vector (Carter, 2001).

Although various vaccine candidates have been tested such as PfS25, PfS28, PfS230 etc, only PfS25 has gone to clinical trials (Wu et al., 2008).

1.6 V

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Traditional vaccines were mostly live or attenuated organism which provided long term sterile immunity and therefore was effective in eliminating many diseases. However dependence of traditional means of preparing vaccines especially one involving live/attenuated organisms is not much popular today because of the ability of the pathogen to revert back to its virulent state.

With mankind facing life threatening diseases, development of improved vaccines with minimal side effects has become a necessity.

1.6.1 DNA VACCINE.

A DNA vaccine is one of the new generation vaccines wherein a plasmid of bacterial DNA encoding the desired antigen under a strong mammalian promoter is inoculated into the individual. The presence of bacterial backbone containing appropriate selection gene makes it easy for the large scale production. The strong CMI response associated with DNA vaccines makes it a very viable technology for producing effective vaccines (Davis et al., 1995). The discovery of DNA vaccines was quite a surprise for the scientific community considering that for direct transfection of cells would be impossible. However studies show that the naked DNA is

16

rapidly taken up by the muscle tissues which is then expressed and presented in context of MHC I (Ulmer et al., 1993). Endogenously synthesized proteins can be then cross-presented by professional antigen presenting cells. This could explain why DNA based vaccines induce strong CTL responses. Overwhelmingly abundant data signifying the viability of DNA based approach in designing vaccines for influenza (Kim and Jacob, 2009), tuberculosis (Hanif et al., 2010), leishmaniasis (Masih et al., 2011), malaria (Hoffman et al., 1997b) etc has been reported.

Inspite of being a good vector for expression of foreign gene, there are potential risks and disadvantages associated with DNA vaccines. They have recently come under renewed scrutiny for a number safety factors such as integration of the DNA into host gene (Wang et al., 2004) and also by their induction of tolerance against the antigen (Mor et al., 1996). When compared with conventional vaccines, DNA vaccines are poor inducers of antibodies (Gramzinski et al., 1998; Polack et al., 2013). A potential risk that could be linked with DNA vaccines are the induction of inflammatory cytokines due to the bacterial DNA backbone of the plasmid construct. Even though such an induction of cytokines can have an adjuvant like effect, too much production can lead to chronic inflammation.

1.6.2 SUBUNIT VACCINE.

Family of these vaccines is not limited to the native recombinant proteins but also incorporates short peptides. Modern technology has facilitated the production of recombinant proteins from various expression systems which closely resemble the native proteins (Hansson et al., 2000).

These vaccines mostly drive the humoral immune responses against the antigen. However the inclusion of adjuvants aids in skew the Th2 response towards a Th1 response. An advantage with these vaccines is the maintenance of conformational stability of the immunodominant epitopes.

Subunit vaccines based on peptides are currently being evaluated for their efficacy against malaria (Mahajan et al., 2010), cancer (Naz and Dabir, 2007) and other diseases. Synthesis of peptides also rules out the potential contamination associated with purification from heterologus systems. Since the peptide vaccine basically involves inoculation with immunodominant B-cell and T-cell, it prevents unnecessary responses directed against other non-protective epitopes

INTRODUCTION

17 (Crowe et al., 2006). For the peptides it becomes essential to couple them with a carrier protein.

One such malaria vaccine was based on the coupling of the central repeat domain of P.falciparum CS protein to the tetanus toxoid (Herrington et al., 1987). However to overcome the dependence on tetanus toxoid Tam and colleagues designed a multiple antigen peptide, in which different branches of the repeat region were synthesized on a polylysine core to form a macromolecular structure (Nardin et al., 1995). In another study a MAP was synthesized by incorporating antigens from different stages of the parasite cycle which generated responses directed against different stages of parasite development (Mahajan et al., 2010).

So despite the ease with which subunit vaccines can be produced most of them require an adjuvant to obtain a strong response.

1.6.2.1 Adjuvants.

Subunit vaccines are poorly immunogenic and therefore require additional components to improve its immunogenicity. These components, known as adjuvants, help to create a favorable niche for the antigen processing and presentation. This can be by enhancing the innate responses which in turn primes an effective adaptive response (McKee et al., 2007; Yuki and Kiyono, 2003). Majority of the adjuvants target the pattern recognition receptors (PRR) for instigating an effective response. One of the earliest adjuvant developed was the Freunds adjuvant, which contained heat killed mycobacteria, signaling via NLR, inflammasome and TLR´s (Freund and Bonanto, 1946). Depending on the adjuvants used, it is possible to skew the response from Th2 to Th1 or the induction of CD8⁺ T-cells instead of CD4⁺ T-cells and vice-versa (Carson and Raz, 1997; Cribbs et al., 2003; Morrow et al., 2010). They can also influence humoral responses by effecting the isotype (Gomez et al., 1998), quantity (Gavin et al., 2006) and avidity (Khurana et al., 2010) of the antibodies produced against the antigen.

Though the number of adjuvants being developed has increased considerably over the last decade, the number of licensed ones for human uses is very limited. In fact the only licensed adjuvant for human purpose is Alum. Despite considerable advances made in immunology development of an effective adjuvant with minimal side effects is still an area of active research.

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One of the main reasons for the slow incorporation of new adjuvants with licensed vaccines is the fear of aggravating the inflammatory responses leading to auto immune disorders (Zandman- Goddard and Shoenfeld, 2005). A recent study about ASO3, a proprietary adjuvant from Glaxo, was reported to increase the incidence of childhood narcolepsy (Nohynek et al., 2012). A variant of this adjuvant, ASO1, is currently used for the RTS,S malaria vaccine.

1.6.3 POXVIRAL VECTORS.

Since the elimination of smallpox by vaccinia virus, use of poxvirus as effective delivery vehicle for heterologus antigens has gained increased popularity. Ability of vector to accommodate large foreign gene combined with the low cost and ease of production makes it an ideal vaccine vector candidate (Pastoret and Vanderplasschen, 2003). Furthermore an effective long lasting humoral and CMI against the heterologus antigen could be developed with these vectors (Smith et al., 1983). Attempts to further enhance the immunogenicity of these vectors gave rise to highly attenuated strains of vaccinia such as Modified Virus Ankara (MVA) and NYVAC. Moreover deletion of immunomodulatory genes from these attenuated strains, such as C6L (Garcia-Arriaza et al., 2011), F1L (Perdiguero et al., 2012) resulted in the production of better vaccine candidates. Earlier studies from our lab also show the importance of the vectors to stimulate mucosal immune cells making them an ideal tool in the fight against diseases which infects via mucosal routes (Gherardi and Esteban, 2005; Gherardi et al., 2003).

An alternative approach to further enhance the immune responses is the prime-boost strategy (Dunachie and Hill, 2003; Ramshaw and Ramsay, 2000). Priming agents such as recombinant DNA, protein, VLP´s, adenoviruses etc when combined with poxvirus are known to induce long lasting immune response (Gomez et al., 2012; Rodriguez et al., 2012; Sanchez-Sampedro et al., 2012). The antigen specific memory CD8⁺ T-cells induced by this approach exhibits an effector phenotype and are highly polyfunctional for IFN-γ, TNF-α and IL-2 (Sanchez-Sampedro et al., 2012). Since an effective malaria vaccine should produce high levels of CD8⁺ T-cells, prime- boost approach is an effective measure for fighting malaria (Li et al., 1993; Schmidt et al., 2010).

In fact many clinical trials based on poxvirus prime-boost in combination with different agents are reported (Table 1).

INTRODUCTION

19 A27 (14K) Vaccinia Protein:

Poxviruses are known to contain many genes that encode for immunogenic proteins such as A27L, A4L, A33, H3L, B5, L1 and many more (Davies et al., 2005; Xiao et al., 2007). Poxvirus antigens have also being used as a strategy to enhance the immunogenic characteristics of other antigens when expressed from viral vectors (Rodriguez et al., 1991). In fact our laboratory has shown that HIV envelope protein fused at the C-terminus with 14K (A27L) or 39K (A4L) protein of vaccinia virus enhanced the immunogenicity of Env, when expressed from the virus vector and inoculated in mice by homologous prime/boost approach, as indicated by an increase in broadly reactive antibodies and CD8⁺ T-cell responses to Env (Collado et al., 2000).

Previous study from our lab has shown the structural organization 14K protein (Vazquez et al., 1998). 14K protein comprises of a structure less region from amino acids 1 to 28 responsible for producing neutralizing antibodies, a helical region from residues 29 to 37, a triple coiled-coil helical region from residues 44 to 72, and a Leu zipper motif at the C terminus. Recently it was reported that amino acid region between 21-32 is responsible for the binding of 14K protein to the heparin sulfates on the cell surfaces, which in turn is aided by the coiled-coil region (Ho et al., 2005).

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Table 1: Prime-boost regimen for malaria vaccine in clinical trials.

Priming Agent Boosting Agent

Immune responses

CD4⁺ CD8⁺ IgG

AdCh63_ME-TRAP MVA_ME-TRAP(Reyes-Sandoval et al., 2010)

- +++ -

FP9-CS MVA-CS(Imoukhuede et al., 2006)

+++ ++ -

DNA-CS MVA-CS(Schneider et al., 1998)

- ++ -

RTS,S/ASO2 MVA-CS(Dunachie et al., 2006)

- + +

OBJECTIVES

23 Development of an efficient vaccine against malaria is a long sought goal of vaccinologist.

Decades of research has finally yielded a vaccine, RTS,S in combination with a powerful adjuvant ASO1E. However, with 50% protection and that too limited for 6 months in children, and with 16.8% after 4 years, this vaccine is far away from being a successful one. With reports suggesting the side effects of adjuvant, the negative factors outweigh its benefits.

The molecular complexity of an antigen is known to influence its immunogenicity. Large oligomeric antigens are more efficient in mounting an effective immune response compared to its monomeric counter parts (Kovacs et al., 2012; Qian et al., 2012). Hence, based on this principle the main objective of this work was to improve the immunogenicity of CS protein based on this principle.

AIMS OF CURRENT STUDY.

Develop a novel way to enhance the immunogenicity of CS protein of Plasmodium by fusing it with the oligomeric A27 (14KDa) vaccinia virus protein (referred to as CS- 14K).

Expression and purification of the fusion protein CS-14K from E.coli.

Physical and biochemical properties of CS-14K.

Characterization of innate properties of CS-14K protein in cell-cultures.

Evaluate the immunological properties (adaptive and memory responses) of CS-14K in mice based on a prime-boost strategy with poxvirus vector MVA expressing CS.

Analyze the efficacy of CS-14K in protecting mice from malaria.

Define immune correlates of protection.

MATERIALS & METHODS

27

3.1 C

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In this study the following cell lines were utilized:

DF-1 (ATCC: CRL-12203)

DF-1 is an immortalized chicken embryo fibroblast cell line. This adherent cell line is mainly used in the propagation of virus. They are grown in Dulbecco´s Modified Eagle Medium supplemented with 2mM of L-Glutamine, 100 μg/ml of streptomycin, 100 IU/ml of penicillin and 10% of heat inactivated fetal calf serum. The cells are maintained at 39ºC with 5% CO₂ and 95% humidity. The medium is changed two times a week and are passaged with trypsin EDTA following which they are subcultivated at 1:5 ratio.

J774 (ATCC: TIB 67)

Mouse macrophage cell line (Balb/C). These adherent cell line exhibit antibody dependent phagocytosis and synthesize large amounts of lysozyme. The cells were maintained in RPMI1640 medium contained L-Glutamine supplemented with 100 μg/ml of streptomycin, 100 IU/ml of penicillin, 50μM β-mercaptoethanol and 10% of heat inactivated fetal calf serum. The cells are maintained at 37ºC with 5% CO2 and 95% humidity. The cells are dislodged using cell scrapper during passages and are subcultivated at a ratio of 1:6.

THP-1 (ATCC: TIB 202)

It is a human monocyte cell line isolated from the peripheral blood from a patient suffering from acute monocytic leukemia. They are grown in suspension and maintained in RPMI 1640 medium containing L-Glutamine supplemented with 100 μg/ml of streptomycin, 100 IU/ml of penicillin, 50μM β-mercaptoethanol and 10% of heat inactivated fetal calf serum. To differentiate into macrophages, cells were treated with 0.5mM PMA (Sigma Aldrich, Spain) overnight.

MATERIALS & METHODS

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3.2 G

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3.2.1 CONSTRUCTION OF VIRUS.

Construction of recombinant MVA virus expressing circumsporozoite protein of P.yoelii 17XNL strain (MVA-CS) has been describe previously (Gonzalez-Aseguinolaza et al., 2003). Briefly the gene encoding the entire CS protein of P.yoelii was isolated from the plasmid, pBS-PY1993, and cloned into the vaccinia insertion vector pJR101, which contains the p7.5 promoter for CS expression and the flanking regions for the HA locus of MVA. The recombinant virus was generated by homologous recombination in DF-1 cells. After several rounds selection based on the expression of B-galactosidase expression the stable recombinant virus was selected.

3.2.2 PURIFICATION OF VIRUS.

Twenty confluent P150 plates of chicken embryo fibroblast, from 11 day old embryonated SPF eggs (Intervet, Spain), was infected with 0.01 PFU/cell of recombinant virus in 5ml of serum free DMEM medium. Following incubation for 1 hour at 37ºC with 5% CO2 the inoculum was removed by aspiration and fresh DMEM medium with 2% FCS was added for 72 hours. After incubation the cells were centrifuged down and washed once with PBS and resuspended in 20 ml of 10 mM Tris-HCl pH9.0. Cells were lysed using sonication (Misonix Sonicator 3000). The sonicated lysate was layered onto 45% sucrose solution and centrifuged in a Beckman SW-28 rotor. The viral pellet was resuspended in 10 mM Tris-HCl pH 9.0 in desired volume.

3.3 G

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pCI-Neo-CS. The PyCSP gene was amplified MVA-CS using the primers CS-XhoI-F (5´-

ACTTACTCGAGATGTGTTACAATGAAGAAAATG-3´) and CS-NotI-R (5´-

ATTGCGGCCGCTTTAAAATATACTTGAAC-3´) to yield a 972 bp fragment lacking the N- terminal signal sequence and C-terminal GPI sequence. The gene was inserted into a mammalian expression vector, pCI-Neo, that had been previously digested with XhoI and NotI followed by SAP treatment (Shrimp Alkaline Phosphatase). The CS gene in both the virus and plasmid were

29 sequenced (Secugen; Spain). The plasmid was purified using Qiagen Mega Prep Kit according to manufacturer’s protocol. Expression of CS from pCI-Neo-CS was confirmed by transfecting DF- 1 cells followed by western blot analysis with CS specific antibodies.

pGEX-CS / pGEX-CS-14K. The CS gene from pCI-Neo-CS plasmid was amplified using the primers CS-EcorI-F (5´-ACTTAGAATTCATGTGTTACAATGAAGAAAATG-3´), CS-NotI-R (5´-ATTGCGGCCGCTTTAAAATATACTTGAAC-3´) for pGEX-CS and with primers CS- EcorI-F and CS-14K-NotI-R (5´-ATTGCGGCCGCTATTAAATATACTTGAAC-3´) for pGEX- CS-14K. The A27L ORF from Vaccinia strain WR (Accession number- YP_233032, www.ncbi.nlm.nih.gov/genbank/) was amplified with the primers A27L-NotI-F (5´-

GCTGCTAGCGGCCGCGAGGCTAAACGCGAAG-3´) and A27L-XhoI-R (5´-

CCCTCGAGTGGGTTACTCATATGGACG-3´) to generate a 276 bp fragment which lacks the first 28 amino acids from the original sequence. The chimeric gene fragment was generated by digesting with Not I followed by ligation. The fusion gene fragment was then inserted into pGEX-6p-1 plasmid to produce pGEX-CS-14K plasmid.

The recombinant plasmid was transformed into DH5α E.coli. The positive clone was selected and purified using Megaprep Kit (Qiagen).

3.4 R

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The recombinant proteins were purified from E.coli strain DH5-α. The starter culture was diluted 1:100 in fresh LB media and allowed to grow at 37ºC till the OD600 reached 0.7, following which IPTG was added to a final concentration of 1 mM. The culture was then incubated in an orbital shaker at 18ºC and 200 rpm for 24 h. After the incubation the cells were harvested and the pellet was suspended in extraction buffer (50 mM Tris-HCl pH 7.5, 250 mM NaCl, 1 mM EDTA and protease inhibitor tablets ROCHE). The cells were then lysed with lysozyme, added to a final concentration of 1mg/ml and incubated on ice for 20 min. Following lysozyme treatment 1%

sarkosyl detergent and 1% triton X-100 was added and incubated at 37ºC for 10 min. Clarified supernatant from the lysis was incubated with Glutathione Sepharose 4B beads at 4ºC overnight.

MATERIALS & METHODS

30

The beads were then washed using three washes with wash buffer I (Extraction buffer with 0.5%

Triton X-114) and three with wash buffer II (Extraction buffer with 0.1% Triton X-114) followed by two washes with extraction buffer. The purified protein was then eluted with 20mM reduced glutathione. The protein was desalted using Amicon centrifugal concentrator and the protein concentration was determined using Bradford reagent. The GST tag from the protein was cleaved using preScission protease according to manufacturer’s protocol (GE Healthcare). The proteins were tested for LPS contamination using chromogenic Limulus Amebocyte Lysate kit (QCL-1000, Lonza) which was maintained below 2 EU per microgram of protein.

3.5 N

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Sera from immunized rabbit were inactivated at 56ºC for 30 min, following which two fold serial dilutions of the serum were made and incubated with 200 PFU of MVA at 37ºC for 1 h.

Afterwards, confluent DF-1 cells were infected in triplicate and were visualized by immunostaining with anti-WR serum after 48 h. As a control, non-immune serum from non immunized animal was used. The number of plaques obtained from each serum dilution was normalized to this control value.

3.6 N

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NO synthesis was measured by estimating the levels of nitrite present in the supernatant. Briefly, J774 cells were stimulated with proteins. For in vivo nitrite analysis, 10⁶ splenocytes from vaccinated animals, sacrificed after 53 days post boost was treated with 5 µg/ml of CS protein.

Nitrite accumulation was measured by treating 50 μl of supernatant with 50 μl of Griess reagent I (1% sulfanilamide solution in 2.4 N HCl) for 10 min in dark followed by the addition of 50 μl of Griess reagent II (0.1% naphthylethlyenediamine in 2.4 N HCl) for 10 min. The assay was read by a spectrophotometer at 540 nm.